PROJECT SUMMARY Loss of sensory hair cells in the inner ear causes irreversible hearing loss in humans. Currently there is no scalable human model system to test pharmacological treatment options for promoting biological restoration of hearing. My laboratory recently developed a novel human inner ear organoid system to generate inner ear sensory epithelia containing functional sensory hair cells from aggregates of human pluripotent stem cells in 3D culture. Although this system is valuable for recapitulating human inner ear development, a human in vitro model of hair cell degeneration is needed for studying hair cell regeneration. We propose to use the inducible Caspase 9 (iCaspase9) genetic ?suicide switch? approach to selectively, rapidly and synchronously ablate hair cells in human inner ear organoids. This system is based on the fusion of human Caspase 9 to a modified human FK-binding protein, allowing for conditional dimerization using a small molecule compound. In Aim 1, we will establish and validate human BRN3C-iCaspase9-tdTomato (BRN3C-iCT) organoids in which sensory hair cells are fluorescently visualized and promptly ablated upon treatment with the dimerizer AP20187. The validated knock-in human embryonic stem cells will then be differentiated into inner ear organoids. After confirmation of hair cell development, AP20187 will be added to culture medium to trigger selective degeneration of sensory hair cells through activation of endogenous Caspase 3. In Aim 2, single-cell RNA- seq will be carried out to detect transcriptional changes in human stem cell-derived sensory hair cells and supporting cells in response to Caspase 9 dimerization. The outcome of this study will provide a paradigm- changing approach for developing and testing chemical compounds capable of promoting hair cell regeneration, and consequently restoring hearing, in individuals with hearing impairments.